The development of labeling dyes that absorb and emit radiation in the near-IR (>600 nm) is important because at those wavelengths interferences from biological species are considerably reduced. The minimized “matrix” effects in the near-IR region result in part because Raman scattering cross-sections are low, and in part because there is reduced absorption and fluorescence from compounds that are typically present in complex biological matrices. In addition, the amount of light that penetrates through tissue typically doubles when the wavelength increases from 550 nm to 630 nm; this intensity doubles again when the wavelength increases from 630 nm to 700 nm, and intensity continues to systematically increase with longer wavelengths throughout the near-IR region. Also, the instrumentation used for detection of near-IR fluorescence is very sensitive with highly efficient single photon detectors, which use avalanche photodiodes and charge-coupled devises (“CCD”). With these sensitive fluorescence detectors one can in some instances detect species at the single-molecule level.
Fluorescence detection in the near-IR region has been demonstrated in a variety of bioanalytical applications: DNA sequencing (see Suzanne J. Lassiter, Wieslaw Stryjewski, Clyde V. Owens, James H. Flanagan, Jr., Robert P. Hammer, Shaheer Khan and Steven A. Soper, “Optimization of sequencing conditions using near-infrared lifetime identification methods in capillary gel electrophoresis,” Electrophoresis 23, 1480-1489 (2002)); detecting DNA restriction fragments; ultra-sensitive analyses for micro-column separations; monitoring the presence of DNA in amplified polymeric chain reactions (“PCR”) products; readout of DNA microarrays (see Yun Wang, Bikas Vaidya, Hannah D. Farquar, Wieslaw Stryjewski, Robert P. Hammer, Robin L. McCarley, Steven A. Soper, Yu-Wei Cheng and Francis Barany, “Microarrays assembled in microfluidic chips fabricated from poly(methyl methacrylate) for the detection of low-abundant DNA mutations,” Anal. Chem. 75, 1130-1140 (2003)); DNA mutation detection (see Norman P. Gerry, Nancy E. Witowski, Joseph Day, Robert P. Hammer, George Barany, and Francis Barany, “Universal DNA Microarray Method for Multiplex Detection of Low Abundance Point Mutations,” J. Mol. Biol., 292, 251-262 (1999)); enzymatic substrate monitoring; and in FRET-based assays (see Musundi B. Wabuyele, Hannah Farquar, Wieslaw Stryjewski, Robert P. Hammer, Steven A. Soper Yu-Wei Cheng, and Francis Barany, “Approaching real-time molecular diagnostics: Single-pair fluorescence resonance energy transfer (spFRET) detection for the analysis of low abundant point mutations in K-ras oncogenes,” J. Am. Chem. Soc. 125, 6937-6945 (2003)).
The major impediment to the use of near-IR fluorescence for various bioanalytical and bioimaging applications has been the lack of chemically robust fluorophores that also are water-soluble, and that exhibit low dark-toxicity. It is desirable that the photophysical properties, as well as the biological properties, of the fluorophores be tunable for particular uses by selectively altering the substituents, the central metal ion, or the axial ligands, while still retaining a very high photon yield. Also, the preferred fluorophore would be the one that would readily conjugate to target biological molecules, such as peptides, nucleic acids and proteins. Finally, when used as a biomarker, a fluorophore must show a high resistance to bleaching. There is an unfilled need for compounds having these properties.
Many commercially available dyes, such as cyanine dyes, are unstable to typical conjugation procedures and conditions, and therefore are not practical for conjugation to biomolecules. A typical commercial dye, such as a tricarbocyanine, exhibits a very low photon yield of approximately 10 photons/molecule.
Phthalocyanines have been used as commercial dyes for almost one hundred years. Pc-derivatives have a well-developed chemistry, and have been used in a wide variety of applications. However, there has been little focus on using phthalocyanines to label biological molecules or for cell staining.
There is an unfulfilled need for chemically robust phthalocyanines and related compounds that can be used in labeling, staining, bioanalytical procedures, including DNA sequencing, and that also can be functionalized to systematically conjugate with biological entities.
J. Flanagan et al., reported that functionalized tricarbocyanine dyes could be used as near-infrared fluorescent probes for biomolecules. Bioconjugate Chem., 8(5): 751-756; J. Flanagan et al., 1998.
Near-infrared, heavy-atom-modified fluorescent dyes for base-calling in DNA-sequencing applications using temporal discrimination have been reported. Anal. Chem. 70 (13): 2676-2684.
U.S. Pat. Nos. 5,135,717 and 5,346,670, and Patent Cooperation Treaty Application WO 90/02747 disclose certain substituted phthalocyanines and related compounds, particularly aluminum phthalocyanines, and their use as dyes in DNA sequencing and other applications.
U.S. Pat. No. 5,494,793 discloses phthalocyanine derivatives monomerically conjugated with an antigen, antibody, oligonucleotide, or nucleic acid, and their use as detectable markers in DNA sequencing and other applications.
European Patent Application 0 502 723 A2 discloses certain tetrazaporphyrins said to be useful for labeling other molecules, and said to be useful in DNA sequencing and other applications.
Patent Cooperation Treaty Application WO 91/18007 discloses a detectably-labeled marker component that comprises a fluorophore moiety comprising a luminescent, substantially planar molecule structure coupled to two solubilizing polyoxyhydrocarbyl moieties, one located on either side of the planar molecular structure. Examples included certain substituted phthalocyanines and triazaporphyrins.
E. A{hacek over (g)}ar et al. “Synthesis and Properties of 1,5-dithio-3-oxa-pentadiyl Bridged Polymeric Phthalocyanines,” Dyes and Pigments 35: 269-278 (1997) discloses certain oxa-thio bridged metal-free and metal phthalocyanine polymers.
Y. Wu et al. “Synthesis and Properties of Soluble Metal-free Phthalocyanines Containing Tetra- or Octa-alkyloxy Substituents,” Dyes and Pigments 37: 317-325 (1998) discloses the synthesis of certain alkyloxy-substituted phthalocyanines.
X. Ding et al. “The Synthesis of Asymmetrically Substituted Amphiphilic Phthalocyanines and Their Gas-sensing Properties,” Dyes and Pigments 40:187-191 (1999) discloses the synthesis of certain asymmetrically substituted amphiphilic phthalocyanines.
I. Rosenthal et al. “The Effect of Substituents on Phthalocyanine Photocytotoxicity,” Photochem. and Photobiol. 46: 959-963 (1987) discloses the testing of several phthalocyanines for photobiological activity.
C. Leznoff et al. “Multisubstituted Phthalonitriles, Naphthalenedicarbonitriles, and Phenanthrenetetracarbonitriles as Precursors for Phthalocyanine Syntheses,” Can. J. Chem. 73: 435-443 (1995) discloses the synthesis of several multisubstituted phthalonitriles, bisaromatic-o-dinitriles, naphthalenedicarbonitriles (5-substituted-2,3-dicyano naphthalenes), and phenanthrenetetracarbonitriles.
C. Leznoff et al. “Synthesis and Photocytotoxicity of Some New Substituted Phthalocyanines,” Photochem. and Photobiol. 49:279-284 (1989) discloses certain ring-substituted phthalocyanines, and their testing for photodynamic activity.
W. Ford et al. “Synthesis and Photochemical Properties of Aluminum, Gallium, Silicon, and Tin Naphthalocyanines,” Inorg. Chem. 31: 3371-3377 (1992) discloses the synthesis of several metal-containing naphthalocyanines said to be relevant in the search for photodynamic therapy agents.
N. Kobayashi, “Optically active ‘adjacent’ type non-centrosymmetrically substituted phthalocyanines,” Chem. Commun. pp. 487-488 (1998) discloses the synthesis of optically active ‘adjacent’ type non-centrosymmetrically substituted phthalocyanines and benzo-substituted phthalocyanines.
N. Kobayashi, et al. “Aggregation, Complexation with Guest Molecules, and Mesomorphism of Amphiphilic Phthalocyanines Having Four- or Eight Tri(ethylene oxide) Chains,” Bull. Chem. Soc. Jpn, 72: 1263-1271 (1999) discloses the synthesis of symmetrical phthalocyanines and zinc phthalocyanines.
N. Brasseur et al. “Synthesis and Photodynamic Activities of Silicon 2,3-Naphthalocyanine Derivatives,” J. Med. Chem. 34: 415-420 (1994) discloses the synthesis of bis(tert-butyldimethylsiloxy)-, bis(dimethylthexylsiloxy)-, bis(tri-n-hexylsiloxy)-, and bis(dimethyloctadecylsiloxy)-silicon 2,3-naphthalocyanines, and their evaluation as potential photosensitizers for photodynamic therapy of cancer.
N. Brasseur et al. “Photodynamic Activities and Skin Photosensitivity of the bis(dimethylthexylsiloxy) silicon 2,3-naphthalocyanine in Mice,” Photochem. and Photobiol. 62: 1058-1065 (1995), discloses complexes that have sensitivity in photodynamic therapy.
D. Terekhov et al. “Synthesis of 2,3,9,10,16,17,23,24-octaalkynylphthalocyanines and the Effects of Concentration and Temperature on Their 1H NMR Spectra,” J. Org. Chem. 61: 3034-3040 (1996) discloses the synthesis of certain octaalkynylphthalocyanines, and discusses the effects that changes in concentration and temperature had on the NMR spectra of these compounds.
A. Cook et al. “Enantiomerically Pure “Winged” Spirane Porphyrazinoctaols,” Angew. Chem. Int. Ed. Engl. 36:760-761 (1997) discloses the synthesis of certain enantiomerically pure porphyrazinoctaols.
A. Montalban et al. “Seco-porphyrazines: Synthetic, structural, and spectroscopic investigations,” J. Org. Chem. 62: 9284-9289 (1997) discloses the synthesis of certain seco-porphyrazines (tetraazaporphyrins).
N. Kobayashi et al., “Phthalocyanines of a novel structure: Dinaphthotetraazaporphyrins with D2h symmetry”, Inorg. Chem. 33:1735-1740 (1994) discloses the synthesis of certain isomerically pure naphthalene molecule-fused tetraazaporphyrins.
N. Mani et al. “Synthesis and characterization of porphyrazinoctamine derivatives: X-ray crystallographic studies of [2,3,7,8,12,13,17,18-octakis(dibenzylamino)-porphyrazinato]magnesium (II) and {2,3,7,8,12,13,17,18-octakis[allyl(benzyl)amino]-porphyrazinato}nickel (II)”, J. Chem. Soc. Chem. Commun. pp. 2095-2096 (1994) discloses the synthesis of certain porphyrazinoctamine derivatives.
W. Sharman et al. “Novel water-soluble phthalocyanines substituted with phosphonate moieties on the benzo rings,” Tetrahedron Letters 37: 5831-5834 (1996) discloses the synthesis of several phthalocyanine derivatives bearing phosphonate substituents directly bound to the aromatic rings of the phthalocyanine, and their possible use as photosensitizers in photodynamic therapy.
M. Brewis et al. “Silicon phthalocyanines with axial dendritic substituents,” Angew. Chem. Int. Ed. 37:1092-1094 (1998) discloses certain silicon phthalocyanines substituted with various axial dendritic substituents.
J. Yang et al. “Synthesis and characterization of a novel octaphenyl substituted solvent soluble phthalocyanine,” J. Heterocyclic Chem. 32: 1521-1524 (1995) discloses the synthesis of certain isomerically pure metal-free or zinc 1,2,3,4,15,16,17,18-octaphenyl-9,10,23,24-tetradodecyloxyphthalocyanines.
J. Young et al. “Synthesis and characterization of di-disubstituted phthalocyanines,” J. Org. Chem. 55: 2155-2159 (1990) discloses the synthesis of certain di-disubstituted phthalocyanines. This paper mentions that the regioselectivity of the usual manner of synthesizing phthalocyanines is poor, and gives mixtures of all possible orientation patterns for the substituents.
T. Baumann et al. “Solitaire-porphyrazines: Synthetic, structural, and spectroscopic investigation of complexes of the novel binucleating norphthalocyanine-2,3-dithiolato ligand,” J. Am. Chem. Soc. 118: 10479-10486 (1996) discloses the synthesis of certain unsymmetrical metalloporphyrazines.
T. Forsyth et al. “A facile and regioselective synthesis of trans-heterofunctionalized porphyrazine derivatives,” J. Org. Chem. 63: 331-336 (1998) discloses the synthesis of regiochemically defined porphyrazines, specifically certain phthalocyanines and derivatives.
E. A{hacek over (g)}ar et al. “Synthesis and spectroscopic investigations of IV-A group phthalocyanines containing macrocycle moieties,” Dyes and Pigments. 36: 407-417 (1998) discloses the synthesis of certain substituted group IV-A (Si, Ge, Sn, and Pb) phthalocyanines.
J. Griffiths et al. “Some observations on the synthesis of polysubstituted zinc phthalocyanine sensitizers for photodynamic therapy,” Dyes and Pigments 33: 65-78 (1997) discloses the syntheses and properties of certain polysubstituted zinc phthalocyanines, and their use as sensitizers for photodynamic therapy.
S. amaz et al. “Synthesis and characterization of new phthalocyanines containing thio-oxa-ether moieties,” Dyes and Pigments. 37: 223-230 (1998) discloses the synthesis of certain metal-free and metal containing-phthalocyanines containing four 9-membered dithiaoxa macrocycles.
D. Ximing et al. “The synthesis and film-forming property of a new amphiphilic phthalocyanine,” Dyes and Pigments 39: 223-229 (1998) discloses the synthesis of an amphiphilic, asymmetrical phthalocyanine.
K. Bello et al. “Some observations on the visible absorption spectra and stability properties of the silicon phthalocyanine system,” Dyes and Pigments 35: 261-267 (1997) discloses the synthesis of a silicon phthalocyanine dye, and discusses its absorption spectra and stability.
N. Kobayashi et al. “Synthesis, spectroscopy, electrochemistry, and spectroelectrochemistry of a zinc phthalocyanine with D2h symmetry,” Chem. Lett. 2031-2034 (1992) discloses a method for the synthesis of certain metallophthalocyanines.
M. Karabork et al. “Synthesis and characterization of phthalocyanines with non-ionic solubilizing groups,” Synth. React. Inorganic Met.-Org. Chem. 1635-1647 (2002) discloses a method for the synthesis of some symmetrical ethylene glycol solubilized metal-free and metallated phthalocyanines
Staining and Labeling
There is an unfilled need for improved methods for effective diagnostic applications, such as labeling or staining biological entities. Further, it is desirable that a family of compounds be available so that a single compound within the family of compounds can be tuned to have specific spectral properties. Such compounds could be used in developing methods for discrete discrimination of particular bases or mutations that may exist in biological samples. However, it is desirable that the differences between compounds within the family of compounds not substantially affect electrophoretic mobility. In addition, it is highly desirable that these compounds be readily conjugated with biological molecules of interest, e.g. to primers or to terminators, and that these compounds, and their conjugate complexes, be able to withstand the thermal and chemical conditions used in immunoassays and other biological assays. We are not aware of any reported compound family that meets these requirements. There is an unfilled need for the development of a suitable family of compounds that satisfies these criteria.
DNA Labeling and Sequencing
Of particular interest are improved methods for efficient DNA analysis. Reported methods use wavelength or color discrimination to differentiate probes or labels. However, it is difficult to use color discrimination to monitor more than 4 probes simultaneously in a single instrument. A major difficulty with color discrimination is the fact that absorption/emission bands tend to be broad, causing difficulties in efficient excitation across the dye set, cross-talk between detection channels due to inefficient filtering, or both—particularly where signal strength is low due to small sample size.
E. Waddell et al. reported “Time-resolved near-IR fluorescence detection in capillary electrophoresis,” J. Liq. Chrom. & Rel. Technol., vol. 23, pp. 1139-1158 (2000).U.S. Pat. No. 5,846,727 discloses a microsystem for rapid DNA sequencing.
Analysis of Mitochondria and Other Cellular Organelles
Of great interest are mitochondria and other cellular organelles. A reduction in the number of mitochondria can be an indication of certain diseases. Mitochondria have a diverse appearance, varying from round-shaped clusters to single-branched formations. Because the mitochondria have different shapes, mitochondrial staining agents are of interest to cell biologists. Methods for the selective identification of mitochondria is important for clinical research, microscopy and monitoring (both in vivo and in vitro analysis).
Several classes of mitochondria-selective stains have previously been reported. Fluorescent aromatic compounds are preferred since fluorescence detection limits are substantially lower than those of conventional light microscopic probes. The most widely used dye for mitochondria staining seems to be Rhodamine 123. The commonly used dyes are described below:
1. Fuchsine derivatives. These dyes are limited in use by the requirement that they be fixed with formalin or osmic acid in 1N HCl, or mercury dichloride, chromate or iron (II or III) salts.
2. Diaminobenzidine (DAB). This thermostable staining method is reversibly inhibited by 0.015M KCN.
3. Styryl dyes. These fluorescent probes have a large Stokes shift and can be used in concentrations 10−8 to 5×10−6 M. However, the time required for uptake of this dye is long, and its quantum efficiency depends on characteristics of the solvent used.
4. Rhodamine and rosamine dyes. There are a number of dyes that are included within this family of dyes:
While the cytotoxicity of Rhodamine 123 was found to be low and the time required for uptake short, this reagent shows low photostability. Treatment with Dinitrophenol (“DNP”) or with high concentrations of ethylene glycol bis(b-aminoethyl-ether)-N,N,N′,N′-tetraacetic acid (“EGTA”) and potassium chloride or sodium azide quench fluorescence. One limitation of this dye is that it is not usually retained in the cells when they are washed.
Fluorescent Mito Tracker Red (chloromethyl X-rosamine, Invitrogen), which is non-toxic in the dark at concentrations 100-250 nM, was found to be light toxic, inducing photosensitizing, depolarization of the mitochondrial membrane and causing apoptosis.
Mito Tracker Orange (chloromethyltetramethylrosamine), another widely used dye, causes mitochondrial depolarization. A chloromethyl group seems to be responsible for keeping the dye associated with the mitochondria via reaction with sulfhydryl reactive groups. The mitochondrial staining ability of both reagents is blocked by any membrane potential altering substance.
MitoTracker Green FM is essentially nonfluorescent in aqueous solutions, but becomes fluorescent when it is deposited in the hydrophobic, lipid environment of mitochondria. This dye accumulates in mitochondria regardless of mitochondrial membrane potential, and it is relatively photostable.
MitoFluor Green and Red Probes (Invitrogen), which also are relatively photostable, do not have chloromethyl moieties and thus are not retained in mitochondria.
5. Carbocyanines. Most carbocyanine dyes stain well when used at low concentrations (˜0.5 μM or ˜0.1 μg/mL), and they are known to be light-toxic. At higher concentrations or when used with cells having high mitochondrial membrane potentials, some representatives of this class (JC-1, Invitrogen) form fluorescent aggregates. The fluorescent emission maximum of these aggregates is shifted about 60 nm from the maximum observed from the non-aggregated species.
6. Nonyl acridine orange. This fluorescent dye stains mitochondria independently of the membrane potential and can be retained up to 10 days. The dye specifically binds to inner membranes, but it is toxic in high concentrations.
7. Avidins. Because biotinylated proteins (biotin carboxylase enzymes) are present almost exclusively in mitochondria, they can be somewhat selectively stained by fluorophore- or enzyme-labeled avidin or streptavidin derivatives. However, unless labeling is performed outside of the cell in vitro, high fluorescence backgrounds are typically observed.
Another example of a cellular organelle is the endoplasmic reticulum (ER), which is the biomolecular factory of the cell. It is here that proteins are made from the genetic information encoded in DNA. New lipids and membranes are also synthesized within the ER system. The ER also plays a role in detoxification of the cell. The ER membrane is chemically very similar to the outer membrane of mitochondria, so stains that label one membrane type will often, but not always, label both types simultaneously.
Stains that are commonly used to label the ER include:
The short chain carbocyanine dye dihexaoxacarbocyanine iodide (DiOC6) labels both the ER and mitochondria. It is highly fluorescent. However, staining can lead to changes in the morphology of the organelles and toxic effects have been noted.
Rhodamine 6G and the hexyl ester of rhodamine B have similar staining characteristics as DiOC6 but with less toxicity and a different fluorescence wavelength.
Long chain versions of DiIC16 (3) (1,1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocyanineperchlorate) and DiIC18(3) (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate have been used to label ER membranes. However, these probes require direct microinjection of the dyes into the cells. This is not a convenient or desirable method of delivery for the dye.
ER-Tracker Blue-White DPX (Invitrogen). Unlike DiOC6, this dye selectively stains the ER and not the mitochondria, and has low toxicity.
ER-Tracker Green and ER-Tracker Red (Invitrogen). These dyes are drug conjugates of glibenclamide BODIPY FL and glibenclamide BODIPY TR, respectively. The glibenclamide moiety binds to sulphonylurea receptors of ATP sensitive potassium channels which are found on the ER. This may lead to variable staining of some cell types due to differential expression of these receptors.
Photodynamic Therapy
Compounds that show light toxicity can be useful as photosensitizers in photodynamic therapy (“PDT”). When cells or living tissues are treated with compounds that become active when exposed to light of a certain wavelength, the cells or tissues can be selectively destroyed.
While not wishing to be bound by this theory, it appears that when an appropriate compound is bound to mitochondria or endoplasmic reticulum in a cell, subsequent exposure of targeted tissue to actinic light produces reactive singlet oxygen in situ, leading to cell death via apoptosis. Unlike necrosis, apoptosis is an orderly process that does not provoke an immune system response. Killing unwanted tissue, e.g., tumor tissue, via apoptosis is preferred over necrosis, as it will generally produce fewer unwanted consequences for the patient. Photodynamic therapy has been used, for example, in treating macular degeneration in the eyes, in treating some skin and throat cancers, and in targeting bacterial and viral pathogens.
M. Hu et al. “Hydroxyphthalocyanines as potential photodynamic agents for cancer therapy,” J. Med. Chem. 41:1789-1802 (1998) discloses several substituted zinc hydroxyphthalocyanines, and their use as possible photodynamic agents for use against cancer. However, it appears that the disclosed PDT agents destroy cells via necrosis, not apoptosis.
Fluorescence resonance energy transfer is a nonradiative process of energy transfer from an excited state fluorophore (donor) to a chromophore (acceptor). An important characteristic of the process is that the energy transfer rate varies inversely with the 6th power of the donor-acceptor separation distance (r6) over the range of 1-10 nm. Thus, by monitoring changes in FRET (e.g. its occurrence or distortion), it is possible to observe changes in the degree of proximity of the FRET fluorophores. FRET-based probes can be used in a variety of in vitro and in vivo DNA-analysis methods such as DNA hybridization detection, PCR monitoring, DNA mutation, cleavage, ligation, recombination detection, RNA synthesis monitoring, as DNA sequencing primers, and for DNA-based biosensors. There is a special interest in developing methods for analyzing complex biological samples using very sensitive FRET-based near-IR detection systems. Those systems would allow multiplexed analysis due to the FRET-capability combined with low interferences from background and availability of the cheap diode lasers for detection in the near-IR.